To determine which viral genes control replication following vaginal transmission of HIV, defined viral constructs must be tested in an animal model. The SIV/rhesus macaque system was used to test the role of the HIV envelope gene in vaginal transmission. SHIVs containing the envelope glycoproteins of two HIV-1 strains, HXB2 and 89.6, in an SIVmac239 backbone have been generated. The HXB2 virus is the prototype of the T-cell tropic viruses. The 89.6 virus is a highly cytopathic variant of the macrophage-tropic viruses. In this study, we demonstrated that both SHIV (HXBc2) and SHIV (89.6) replicate following intravenous injection of an inoculum containing a mixture of both viruses. At the CRPRC, two male rhesus macaques were inoculated with a mixture of HXB2 SHIV clone and SHIV (89.6). Ten clones were produced from PBMC of each animal taken at 2 weeks PI and the env gene of each clone was sequenced. Approximately half the clones had the 89.6 sequence and half had the HXB sequence. Thus, both SHIV (HXBc2) and SHIV (89.6) replicate following intravenous injection of an inoculum containing a mixture of both viruses. Two mature female rhesus macaques were inoculated intravaginally 4 times (twice a week for 2 weeks) with a mixture of HXB2 SHIV clone and SHIV (89.6). Ten clones were produced from PBMC of each animal taken at 2 weeks PI and the env gene of each clone was sequenced. All the clones had the 89.6 sequence. These results demonstrate vaginal transmission of SHIVs for the first time and that the viral env gene confers the ability to initiate infection via the vaginal route. *KEY*Viral determinants, Mucosal transmission